小鼠单个植入前胚胎cDNA文库的构建及应用

哺乳动物合子基因组激活和植入前胚胎阶段特异性表达基因的研究一直是发育生物学研究的重点。发现和克隆植入前胚胎阶段特异性表达的基因需要有效的方法,阶段特异性cDNA文库的构建和筛选是一种较好的方法。用小鼠单个成熟卵细胞、受精卵、2细胞、4细胞和8细胞胚胎分别构建了cDNA文库。滴度分别为:62×105、83×105、104×106、151×106和162×106。随机挑选了29个克隆并进行了序列分析,结果表明,和已知表达序列标签(ESTs)同源的序列为6552%(1929),未知序列为1379%(429),并发现了2个重复序列。用特异性引物,分别对小鼠持家基因(βactin)和发育特异基因(OCT4)进行PCR扩增,结果表明,所建立的cDNA文库可以反映小鼠胚胎在不同发育阶段整个基因群体的转录活性,为阶段特异性基因的筛选和小鼠早期阶段基因表达模式的研究提供了一种有价值的资源库 。     

山羊β—酪蛋白基因启动子指导人血清白蛋白基因在小鼠组织中的特异性表达

对本所构建的pcDNA3.1-β6.7hALBm表达载体行尾静脉注射直接转染哺乳期母鼠的活体组织。通过RT－PCR检测hALBm基因在小鼠几种组织中的表达来研究该构建体的组织特异性。结果：构建体在受试鼠组织中的表达显示了较好的乳腺组织特异性。说明构建具有较长5'端上游乳蛋白调控区的乳腺特异性表达载体对组织特异性表达是必需的。     

水稻胚乳特异性启动子的克隆及序列分析

目的:克隆获得水稻胚乳特异表达启动子pGluB-1。方法:采用PCR方法从水稻\"台粳9号\"基因组DNA中扩增出pGluB-1启动子序列,并克隆至pMD20-T载体上,酶切鉴定后进行测序并对测序结果进行生物信息学分析。结果:获得大小为1 353bp的pGluB-1启动子序列,该序列与已报道序列的同源性为97%。启动子功能预测及顺式作用元件分析表晨所克隆的启动子序列含有ACGT基序、AACA基序、GCN4基序和醇溶蛋白框等胚乳特异表达必需的调控元件,其序列差异可能是由于不同品种个体差异及多态性的影响。结论:成功克隆出pGluB-1启动子,为实现外源目的基因在水稻胚乳中特异性表达奠定了实验基础。     

Promoters maintain their relative activity levels under different growth conditions

Most genes change expression levels across conditions, but it is unclear which of these changes represents specific regulation and what determines their quantitative degree. Here, we accurately measured activities of ～900 S. cerevisiae and ～1800 E. coli promoters using fluorescent reporters. We show that in both organisms 60–90% of promoters change their expression between conditions by a constant global scaling factor that depends only on the conditions and not on the promoter's identity. Quantifying such global effects allows precise characterization of specific regulation—promoters deviating from the global scale line. These are organized into few functionally related groups that also adhere to scale lines and preserve their relative activities across conditions. Thus, only several scaling factors suffice to accurately describe genome‐wide expression profiles across conditions. We present a parameter‐free passive resource allocation model that quantitatively accounts for the global scaling factors. It suggests that many changes in expression across conditions result from global effects and not specific regulation, and provides means for quantitative interpretation of expression profiles.     

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNa polymerase III

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to -amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.     

Gametophytic expression of genes controlling endosperm development in maize

Summary An indirect approach was adopted to select viable mutants affecting the male gametophytic generation in maize. This approach consists of a selection of endosperm defective mutants followed by a test for gametophytic gene expression, based on the distortion from mendelian segregation and on the measurement of pollen size and pollen sterility. The material used consisted of 34 endosperm defective viable mutants introgressed in B37 genetic background. Complementation tests indicated that the mutation in the collection of mutants affected different genes controlling endosperm development. The study of the segregation in F2 revealed four classes of de (defective endosperm) mutants: (1) mutants in which the mutation does not affect either gametophytic development or function; (2) mutants in which the effect on the gametophyte affects pollen development processes; (3) mutants showing effects on both pollen development and function, and (4) mutants where only pollen tube growth rate is affected. Positive and negative interactions between pollen and style were detected by means of mixed pollination (pollen produced by de/de plants and pollen from an inbred line used as a standard and carrying genes for colored aleurone), on de/de and de/ + plants. Positive interactions were interpreted as methabolic complementation between defective pollen and normal styles.     

Transmission ratio distortion at the growth hormone gene (GH1) in bovine preimplantation embryos: An in vitro culture‐induced phenomenon?

The growth hormone gene (GH1) and its polypeptide product (GH) have a crucial role in reproduction, embryogenesis and general development. A polymorphism present in the fifth exon of the bovine GH1 gene (GH1 p.Leu127Val) has been associated with GH release and milk production in cattle. The objective of the present study was to examine the genotype frequencies of the GH1 p.Leu127Val polymorphism in bovine blastocysts produced in vitro and in vivo to determine if allelic variation of the GH1 gene affects embryo development and survival. A heterozygous (p.Leu127/Val127) sire was used for in vitro fertilization of oocytes of unknown maternal genotype (n = 104) and known maternal genotype (n = 115). PCR amplification and genotyping of the GH1 gene from Day 8 blastocysts derived from these fertilized oocytes demonstrated that there was significant over-representation from the expected Mendelian ratio of GH1 p.Leu127/Leu127 homozygotes from oocytes of known maternal genotype (P = 0.006). Contrary to this, analysis of in vivo-produced bovine blastocysts of known parental GH1 genotype (n = 69) did not reveal an overrepresentation of GH1 p.Leu127/Leu127 homozygotes. These results suggest that developing in vitro-produced embryos are exposed to a selection process, probably due to a less favorable culture environment, that acts to increase the number of GH1 p.Leu127/Leu127 homozygotes, thereby giving rise to the observed transmission ratio distortion (TRD) of GH1 genotypes when compared to in vivo produced embryos.     

华山新麦草胚和胚乳的发育研究

采用常规石蜡切片法,观察了华山新麦草胚和胚乳的发育过程,结果表明,华山新麦草胚和胚乳的发育过程与一般禾本科植物基本相同,胚胎发生属紫宛型,顶细胞和基都参与胚体的形成,胚胎发育经过二细胞原胚,多细胞原胚,球形原胚,梨形原胚,分化胚和成熟胚阶段,成熟胚具有胚根,胚芽,盾片,胚牙鞘,胚根鞘,外胚叶等典型禾本科植物成熟胚的结构,胚乳发育类型为核型,包括游离核阶段,细胞化阶段和生长成熟阶段,待大量游离核形成之后才形成细胞壁,紧贴胚囊的一层胚乳细胞最后形成种子的糊粉层,其余的胚乳细胞最后充满淀粉粒,其特点为：（1）有双球形原胚的现象;（2）反足细胞解体较早;（3）胚乳游离核时期和细胞时期胚乳细胞核的核仁多样。     

ZmEBE genes show a novel,continuous expression pattern in the central cell before fertilization and in specific domains of the resulting endosperm after fertilization

Two novel maize genes expressed specifically in the central cell of the female gametophyte and in two compartments of the endosperm (the basal endosperm transfer layer and the embryo surrounding region) were characterized. The ZmEBE (embryo sac/basal endosperm transfer layer/embryo surrounding region) genes were isolated by a differential display between the upper and the lower half of the kernel at 7 days after pollination (DAP). Sequence analysis revealed ORFs coding for two closely related proteins of 304 amino acids (ZmEBE-1) and 286 amino acids (ZmEBE-2). This size difference was due to differences in the splicing of the two genes. Both protein sequences showed significant similarity to the DUF239 family of Arabidopsis, a group of 22 proteins of unknown function, a small number of which are putative peptidases. ZmEBE genes had a novel cell type-specific expression pattern in the central cell before and the resulting endosperm after fertilization. RT-PCR analysis showed that the expression of both genes started before pollination in the central cell and continued in the kernel up to 20 DAP with a peak at 7 DAP. In situ hybridization revealed that the expression in the kernel was restricted to the basal transfer cell layer and the embryo surrounding region of the endosperm. The expression of ZmEBE-1 was at least 10 times lower than that of ZmEBE-2. Similarly to other genes expressed in the endosperm, ZmEBE-1 expression was subject to a parent-of-origin effect, while no such effect was detected in ZmEBE-2. Sequence analysis of upstream regions revealed a potential cis element of 33 bp repeated 7 times in ZmEBE-1 and ZmEBE-2 between positions -900 and -100. The 1.6 kb ZmEBE-2 upstream sequence containing the seven R7 elements was able to confer expression in the basal endosperm to a Gus reporter gene. These data indicate that ZmEBE is potentially involved in the early development of specialized domains of the endosperm and that this process is possibly already initiated in the central cell, which is at the origin of the endosperm.     

番茄rbcS3A启动子控制的GUS融合基因在转基因水稻中的表达

为研究不同启动子用于转基因水稻,克隆了番茄Rubisco小亚基rbcS3A基因的5′上游调控区,构建了由rbcS3A启动子引导的GUS嵌合基因,并经农杆菌介导导入到水稻中。对转基因水稻植株中GUS活性的定性与定量测定结果表明,rbcS3A启动子可驱动GUS报告基因在转基因水稻植株茎和叶组织中高效表达,而在根和种子等器官中不表达或表达活性极弱,表现出一定的组织特异性。在转基因水稻中,番茄rbcS3A启动子驱动外源基因的表达不受光诱导。     

Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive–negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a , one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T₀) transgenic knock-in plants obtained were found to carry only one copy of GUS , with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter -fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive–negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.     

何首乌胚和胚乳的发育

何首乌为直生胚珠,双珠被。胚发育属于柳叶菜型。心形胚柄最为发达,鱼雷形胚期胚柄奶化。早期胚胎发育营养的主要来源可能是合了中积累的淀粉和胚柄吸收来的营养。成熟胚中积累了大量的蛋白质和淀粉粒。胚乳发育属核型。从球形胚期起,胚乳细胞化过程由珠孔端向合点端逐渐推进。初始垂周壁源于姊妹核间的细胞反或非姊妹核间由次生成膜生的细胞板。初始平周壁源于有丝分裂所产生的细胞板。心形胚期,除合点端保持游离核胚乳吸器外,     

特异性启动子在植物基因工程中的应用

选择特异性启动子构建植物表达载体,是实现基因表达三维调控的重要策略,并已应用于植物品质改良基因工程、抗性基因工程及植物生物反应器等领域。文章综述了特异性启动子的结构、类型、研究方法及在植物基因工程研究中的应用进展和发展前景。